Summary This Project is directed toward the induction of xenotransplantation tolerance through mixed hematopoietic chimerism, which is to date the only treatment regimen that has been successful in translating allogeneic tolerance induction from animal models to the clinic. Prior to the current project period, attempts to achieve mixed chimerism across the pig-to-primate barrier had failed due to rapid clearance of infused porcine cells from the circulation of primates. During this period, we have tested the hypothesis that this rapid clearance might be due to the absence of the primate analog of the species-specific cell-membrane protein CD47 on the surface of porcine cells leading to rapid clearance of pig cells (PBSC) by recipient macrophages. We have produced and tested mobilized peripheral blood stem cells from human CD47 (hCD47) transgenic GalT-KO miniature swine for their ability to induce chimerism in baboons. Our results demonstrated markedly increased survival of porcine cells injected into baboons that was dependent on the level of hCD47 expression on donor cells. Furthermore, recipients of hCD47-expressing swine PBSC demonstrated prolonged survival of donor swine skin xenografts in the absence of immunosuppression. In the current proposal, we will attempt to increase the level and duration of porcine mixed chimerism by: 1) Confirming the effect of high expression of the hCD47 transgene in porcine hematopoietic stem cells (HSC) and the use of intra-bone HSC inoculation on the establishment of chimerism and prolonged pig skin graft survival in baboons; 2) Testing the effect of ex-vivo expanded recipient Tregs on enhancement of xenogeneic chimerism and engraftment of hCD47/GalT-KO HSC; and 3) Testing the effect of new transgenes in genetically engineered swine, produced in Project 4, that are designed to increase the level and duration of donor chimerism and induce tolerance to skin grafts. In addition to advancing our goal toward the induction of xenograft tolerance, the experiments planned will provide basic information on xenogeneic stem cell engraftment and on the immunologic pathways responsible for xenogeneic rejection and tolerance induction in primates. These studies should therefore have both theoretical and practical implications for the eventual application of xenotransplantation as a clinical modality.